Substrate Deconstruction and the Nonadditivity of Enzyme Recognition

Predicting substrates for enzymes of unknown function is a major postgenomic challenge. Substrate discovery, like inhibitor discovery, is constrained by our ability to explore chemotypes; it would be expanded by orders of magnitude if reactive sites could be probed with fragments rather than fully elaborated substrates, as is done for inhibitor discovery. To explore the feasibility of this approach, substrates of six enzymes from three different superfamilies were deconstructed into 41 overlapping fragments that were tested for activity or binding. Surprisingly, even those fragments containing the key reactive group had little activity, and most fragments did not bind measurably, until they captured most of the substrate features. Removing a single atom from a recognized substrate could often reduce catalytic recognition by 6 log-orders. To explore recognition at atomic resolution, the structures of three fragment complexes of the β-lactamase substrate cephalothin were determined by X-ray crystallography. Substrate discovery may be difficult to reduce to the fragment level, with implications for function discovery and for the tolerance of enzymes to metabolite promiscuity. Pragmatically, this study supports the development of libraries of fully elaborated metabolites as probes for enzyme function, which currently do not exist

Structure of the Trehalose-6-phosphate Phosphatase from <i>Brugia malayi</i> Reveals Key Design Principles for Anthelmintic Drugs

<div><p>Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from <i>Brugia malayi</i>. In the model nematode <i>Caenorhabditis elegans</i>, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in <i>Mycobacterium tuberculosis</i>. We determined the X-ray crystal structure of T6PP from <i>B. malayi</i>. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.</p></div

G6PC mRNA Therapy Positively Regulates Fasting Blood Glucose and Decreases Liver Abnormalities in a Mouse Model of Glycogen Storage Disease 1a

International audienceGlycogen storage disease type Ia (GSD1a) is an inherited metabolic disorder caused by the deficiency of glucose-6-phosphatase (G6Pase). GSD1a is associated with life-threatening hypoglycemia and long-term liver and renal complications. We examined the efficacy of mRNA-encoding human G6Pase in a liver-specific G6Pase-/- mouse model (L-G6PC-/-) that exhibits the same hepatic biomarkers associated with GSD1a patients, such as fasting hypoglycemia, and elevated levels of hepatic glucose-6-phosphate (G6P), glycogen, and triglycerides. We show that a single systemic injection of wild-type or native human G6PC mRNA results in significant improvements in fasting blood glucose levels for up to 7 days post-dose. These changes were associated with significant reductions in liver mass, hepatic G6P, glycogen, and triglycerides. In addition, an engineered protein variant of human G6Pase, designed for increased duration of expression, showed superior efficacy to the wild-type sequence by maintaining improved fasting blood glucose levels and reductions in liver mass for up to 12 days post-dose. Our results demonstrate for the first time the effectiveness of mRNA therapy as a potential treatment in reversing the hepatic abnormalities associated with GSD1a

Proposed model of trehalose 6-phosphate in the active site of T6PP.

<p>The FTMap server was used to identify hot spots where protein-substrate interactions may occur. Analysis of the T6PP enzyme from <i>T. acidophilium</i> (1U02) (A), and <i>B.</i> malayi (B) reveal hot spots near the interface of the cap and core domains. These hot spots are cradled by the structurally conserved C1-Loop. T6P was placed manually into the active site of T6PP by coordinating the Mg²⁺ cation with the phosphate group (C). The residues identified as important via mutagenesis and kinetics are labeled and can be seen in proximity to the trehalose moiety.</p

Comparison of ligand binding between sucrose-6-phosphate phosphatase and trehalose-6-phosphate phosphatase.

<p>An analysis of the crystal structure of the S6PP-sucrose 6-phosphate (S6P) complex (PDB: 1U2T) and the model of the T6PP-trehalose 6-phosphate (T6P) complex revealed that the stereochemistry of the glycosidic bond might affect specificity. The cap can be found in a different position in the S6PP-S6P complex than either the crystal structure or cap-closed model of T6PP, affecting the size and shape of the active site cavity. An extensive hydrogen-bonding network exists between S6PP and S6P (black dashed lines), utilizing residues from both the cap domain (orange) and the core domain (blue) (A). Positioning of the cap in S6P may be impacted by the α(1→2)β glycosidic bond of sucrose 6-phosphate versus the α(1→1)α glycosidic bond in trehalose 6-phosphate. An overlay of T6P from our model and S6P reveals a putative clash between S6P and residues 254–265 (light grey loop) in T6PP (B) potentially explaining the lack of turnover or binding of S6P.</p

Putative substrate interacting residues in <i>B. malayi</i> analyzed by mutagenesis and kinetics.

<p>Residues in the cap and core regions analyzed by mutagenesis are depicted as sticks and colored according to kinetic parameters: no effect on kinetic parameters (grey); catalytically inactive (blue); decreases in k_cat (green); significant changes in k_cat and K_I for a T6S substrate analog (red).</p

Two binding pockets in the T6PP enzyme.

<p>Analysis of the hypothesized binding model for trehalose 6-phosphate and enzyme kinetics suggests that inhibitors should interact with two pockets in order to maximize interactions. Trehalose 6-phosphate (A) and trehalose 6-sulfate (B) presumably bind in the phosphoryl-binding and sugar-binding pockets while glucose 6-phosphate (C) and trehalose (D) interact in only one pocket.</p

Steady-state kinetic constants and dissociation constants for inhibitors for wild-type and variant <i>B. malayi</i> T6PP-catalyzed hydrolysis of trehalose 6-phosphate.

a<p>NA represents no activity detected above detection limit.</p